The potential applications for biologically active proteins have greatly increased. Commercial implementation of this technology now frequently depends on the ability to isolate these substances at reasonable cost. Until recently, separation technology which could support industrial applications was limited to filtration and centrifugation. However, these techniques are extremely dependant upon particle size and therefore approach their limit of usefulness during the harvest of even small intact microorganisms. The problems encountered are therefore greatly increased during the attempted isolation of intracellular components from ruptured cells where component size is, of course, greatly reduced.
The process of affinity partitioning using two phase aqueous systems has been suggested for some separations. Affinity partitioning basically involves the formation of multiple, distinct phases in a common solvent following the addition of materials, such as polymers, which produce immiscible phases when in solution, and the selective affinity of a molecule for one phase over the other.
Aqueous two phase systems have been known since the late nineteenth century from the work of Beijerinck who published has findings regarding aqueous phase formation with agar and gelatin. As affinity partitioning is not dependant upon particle size as are conventional techniques such as filtration and centrifugations it offers the potential of improved recovery of cellular components. The use of affinity partitioning in the isolation of enzymes from other cellular matter is disclosed in U.S. Pat. No. 4,144,130. Affinity partitioning technology has further been employed to date in the recovery of interferon (U.S. Pat. No. 4,343,735), the isolation of human coagulation factors VII and VIIa (U.S. Pat. No. 4,470,969) and the isolation of deoxyribonucleic acid (U.S. Pat. No. 4,207,200).
A number of systems suitable for the separation of enzymes are known, for instance those described in Albertson, P. A., Partition of Cell Particles and Macromolecules, Uppsala, 1st edition (1960), 2nd edition (1971) and U.S. Pat. No. 3,897,414. The aqueous systems most commonly employed are those consisting of polyethylene glycol/dextran or polyethylene/glycol/water-soluble inorganic salt.
Due to the relatively low concentration of enzyme in the feed liquors, the relatively low efficiencies at which these processes may operate and the large quantities of product desired, commercial application of two phase aqueous system technology requires the use of large volumes of the phase-forming liquids. Recycle of these liquids would therefore greatly increase the commercial viability of this technology. However, the isolation of product from a system and subsequent recycle of liquids must be accomplished without adversely effecting the properties of the material which is partitioned in the system. For instance, the activity of enzymes isolated through use of multi-phase systems should not be adversely affected to an undue degree. Moreover, isolation of material from the system's phase-forming liquids should be accomplished in a manner such that neither the liquids nor the isolated material require extensive processing to place it in a form which can be employed in its final application.
The applicants have therefor sought to provide through the present invention a method for recycle of the phase-forming liquids of an aqueous system having at least two aqueous phases and containing biological material, such as enzymes, through the introduction of a water-insoluble inert binder material onto which biological material, such as enzymes, may be adsorbed. The binder with its adsorbed material may then be readily recovered through conventional techniques, such as filtration or centrifugation, leaving the liquid available for recycle. The biological material may be released from the binder or, if its final application allows, it may be used in this combined form.